Stomach submucosa derived tissue graft

ABSTRACT

A tissue graft composition comprising stomach submucosal tissue delaminated from both the luminal portion of the tunica mucosa and the smooth muscle layers of the muscularis externa of a stomach of a warm blooded vertebrate is described. The graft composition can be or implanted into a host to replace or support damaged or diseased tissues.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a U.S. national application of internationalapplication Ser. No. PCT/US97/23010 filed Dec. 10, 1997, which claimspriority to U.S. Provisional application Ser. No. 60/032,683 filed Dec.10, 1996.

FIELD OF THE INVENTION

The present invention relates to a tissue graft composition and methodsfor its preparation and use. More particularly, the present invention isdirected to non-immunogenic tissue graft compositions comprising stomachsubmucosa and use of same to promote endogenous tissue growth.

BACKGROUND AND SUMMARY OF THE INVENTION

It is known that compositions comprising the tunica submucosa of theintestine of warm-blooded vertebrates can be used advantageously astissue graft materials. See U.S. Pat. Nos. 4,902,508 and 5,281,422. Thetissue graft compositions described in those patents are characterizedby excellent mechanical properties, including a high burst pressure, andan effective porosity index which allows such compositions to be usedbeneficially for vascular graft and connective tissue graft constructs.When used in such applications the graft constructs appear not only toserve as a matrix for the regrowth of the tissues replaced by the graftconstructs, but, indeed, promote or induce such regrowth of endogenoustissue. Common events to this remodeling process include: widespread andvery rapid neovascularization, proliferation of granulation mesenchymalcells, biodegradation/resorption of implanted intestinal submucosaltissue material, and lack of immune rejection.

It is also known that intestinal submucosa can be fluidized bycomminuting and/or enzymatic digestion, without loss of its apparentbiotropic properties, for use in less invasive methods of administration(e.g., by injection or topical application) to host tissues in need ofrepair. See U.S. Pat. No. 5,275,826.

There has been much additional research effort directed to finding othernatural and synthetic materials having the requisite properties for useas tissue grafts. Surprisingly, it has been found that stomach submucosaprepared by delamination of stomach tissue of warm-blooded vertebratesexhibits mechanical and biotropic properties similar to that which hasbeen reported for intestinal submucosal tissue. It can be substitutedfor intestinal submucosa tissue in most, if not all, of the applicationspreviously reported for intestinal submucosa.

The tissue graft composition of the present invention comprises stomachsubmucosa derived from stomach tissue of a warm-blooded vertebrate. Thewall of the stomach is composed of the following layers: the tunicamucosa (including an epithelium layer, a tunica propria layer consistingof reticular or fine areolar tissue, and a glandular layer), the tunicasubmucosa layer (composed of areolar tissue and lacking glands), thetunica muscularis layer (composed of three layers of muscle), and theserosa (a layer of mesothelium outside the loose connective tissue whichinvests the muscle layers). Blood vessels, lymphatic tissue andneurological tissue also pervade the stomach tissues including thetunica submucosa.

Stomach submucosal tissue in accordance with the present inventioncompises stomach submucosa delarminated from the glandular portion ofthe tunica mucosa and the smooth muscle layers of the muscularisexterna. The composition has proven to have the ability to induceconnective tissue remodeling and wound healing in a fashion very similarto that of intestinal submucosa as described in U.S. Pat. No. 5,275,826.Specifically, the stomach submucosa composition causes cellproliferation in vitro, supports cell growth when used as a growthsubstrate material, and induces the formation and repair of connectivetissue structures such as Achilles tendon when placed in xenogeneic hostspecies. Stomach submucosa appears to be non-antigenic, and inducesresponses in vivo that are recognized components of wound healing suchas neovascularization, cellular infiltration, deposition ofextracellular matrix, and eventual degradation and replacement of theimplanted material with host tissues. The present graft composition canbe implanted or injected into a vertebrate host to induce the repair orreplacement of damaged or defective tissues.

DETAILED DESCRIPTION OF THE INVENTION

The tissue graft composition in accordance with the present inventioncomprises stomach submucosa of a warm-blooded vertebrate delaminatedfrom adjacent stomach tissue layers. The present tissue graftcomposition thus comprises the stomach submucosa delaminated from thesmooth muscle layers of the muscularis externa and at least the luminalportion of the mucosal layer of a segment of the stomach of awarm-blooded vertebrate. In one embodiment, the stomach submucosaltissue compositions comprise the tunica submucosa and basilar portionsof the tunica mucosa of the stomach of a warm blooded vertebrate.Typically the delamination technique described below provides a tissuecomposition consisting essentially of stomach submucosa. Thosecompositions are referred to herein generically as stomach submucosaltissue.

The glycosaminoglycan (GAG) content of stomach submucosa has beenanalyzed. The types of GAGs identified in stomach submucosa includeheparin, chondroitin sulfate A, chondroitin sulfate B, and hyaluronicacid. In contrast to intestinal submucosa, heparan sulfate was not foundin stomach submucosa.

The amount of water present in a sample of stomach submucosa, variesdependent upon the processing steps utilized after the delamination ofthe tissue. In accordance with one procedure the material was left to"drain" itself of water by sitting on a solid surface for ten minutes.The weight of the material was then determined, and the material wassubjected to a mechanical wringer to remove additional excess waterbefore freezing and lyophilization. Repeated measurements on twoseparate specimens showed a water content which ranged from about 80% toabout 94%. The degree of variability is undoubtedly due to thevariability of determining the initial "wet weight". However, it can besafely said that the water content of stomach submucosa is between 80and 95%.

The stomach submucosa composition of this invention is typicallyprepared from stomach tissue harvested from animals raised for meatproduction, including, for example, pigs, cattle and sheep or otherwarm-blooded vertebrates. This tissue is normally a discarded by-productof meat processing. Thus, there is an inexpensive commercial source ofstomach tissue for use in preparation of the tissue compositions inaccordance with the present invention.

The preparation of stomach submucosa from a segment of stomach issimilar to the procedure for preparing intestinal submucosa detailed inU.S. Pat. No. 4,902,508, the disclosure of which is expresslyincorporated herein by reference. A segment of stomach tissue is firstsubjected to abrasion using a longitudinal wiping motion to remove theouter layers (particularly the smooth muscle layers) and the luminalportions of the tunica mucosa layers. The resulting submucosa tissue hasa thickness of about 100 to about 200 micrometers, and consistsprimarily (greater than 98%) of acellular, eosinophilic staining (H&Estain) extracellular matrix material. Occasional blood vessels andspindle cells consistent with fibrocytes are scattered randomlythroughout the tissue. Typically the submucosa is rinsed with water forapproximately 2 hours and optionally stored in a frozen hydrated stateuntil used as described below. Delamination of the tunica submucosa fromboth the muscularis externa and at least the luminal portions of thetunica mucosa layers and rinsing of the submucosa provides an acellularstomach submucosal tissue matrix.

Fluidized stomach submucosa can be prepared in a manner similar to thepreparation of fluidized intestinal submucosa, as described in U.S. Pat.No. 5,275,826 the disclosure of which is expressly incorporated hereinby reference. The submucosa tissue is comminuted by tearing, cutting,grinding, shearing and the like. Grinding the submucosa in a frozen orfreeze-dried state is preferred although good results can be obtained aswell by subjecting a suspension of submucosa pieces to treatment in ahigh speed (high shear) blender and dewatering by centrifuging anddecanting excess water. Additionally, the conmuinuted fluidized tissuecan be solubilized by enzymatic digestion of the stomach submucosaincluding the use of proteases, such as trypsin or pepsin, or otherappropriate enzymes such as a collagenase or a glycosaminoglycanase, orthe use of a mixture of enzymes, for a period of time sufficient tosolubilize said tissue and form a substantially homogeneous solution.

The present invention also contemplates the use of powder forms ofstomach submucosa. In one embodiment a powder form of stomach submucosais prepared by pulverizing stomach submucosal tissue under liquidnitrogen to produce particles ranging in size from 0.1 to 1 mm². Theparticulate composition is then lyophilized overnight and sterilized toform a solid substantially anhydrous particulate composite.Alternatively, a powder form of stomach submucosa can be formed fromfluidized submucosa by drying the suspensions or solutions of comminutedand/or partially digested stomach submucosa.

The stomach submucosal tissue compositions of the present invention lendthemselves to a wide variety of surgical applications relating to therepair or replacement of damaged tissues, including, for example therepair of vascular and connective tissues. Connective tissues for thepurposes of the present invention includes bone, cartilage, muscle,tendons, ligaments, and fibrous tissue including the dermal layer ofskin.

In accordance with the present invention, stomach submucosal tissue isused to prepare tissue graft compositions that are useful for inducingthe formation of endogenous tissue at a desired site in a warm bloodedvertebrate. Compositions comprising stomach submucosa can beadministered to a vertebrate host in an amount effective to induceendogenous tissue growth at a site in the host in need of same due tothe presence of damaged or diseased tissue. The stomach submucosacompositions can be administered to the host in either solid or sheetform, by surgical implantation alone or in combination with otherart-recognized implant compositions, or in fluidized form, by injection.

In one embodiment the present stomach submucosa compositions in sheetform can be used to form vascular grafts. The diameter of the graftshould be about the same as the diameter of the recipient blood vessel.This is accomplished by manipulating the stomach submucosa to define acylinder having diameter approximately the same as that of the recipientblood vessel and suturing or otherwise securing the tissue graftlongitudinally to form said vascular graft. Thus, for example, avascular graft can be prepared by selecting a sterile glass rod havingan outer diameter equal to that of the recipient blood vessel, wrappingthe stomach submucosa sheet around the glass rod and gathering theredundant tissue. The desired lumen diameter is achieved by suturingalong the length of the graft (for example, using two continuous suturelines or a simple interrupted suture line) or by using otherart-recognized tissue securing techniques. The vascular graft issurgically substituted for a damaged or diseased blood vessel usingstandard vascular surgery techniques.

Consistent with the use of stomach submucosa as a vascular graftmaterial, stomach submucosa possesses mechanical properties similar tothose of intestinal submucosa and highly desirable for such tissue graftmaterials, including low porosity index and a high burst pressure. Thoseskilled in the art will appreciate that vascular graft material must beof low enough porosity to prevent intraoperative hemorrhage and yet ofhigh enough porosity to allow extension of a newly-developed vasavasorum through the graft material to nourish the luminal surface.

The present stomach submucosa segments can also be used in accordancewith this invention as a tissue graft construct for use in the repair orreplacement of connective tissues using the same procedures describedfor use of intestinal submucosa in U.S. Pat. Nos. 5,281,422 and5,352,463, the disclosures of which are expressly incorporated herein byreference. The stomach submucosa composition can be used in itsdelaminated natural sheet form or it can be cut longitudinally orlaterally to form elongated tissue segments. Such segments have anintermediate portion, and opposite end portions and opposite lateralportions which can be formed for surgical attachment to existingphysiological structures, using surgically acceptable techniques.

The grafts formed and used in accordance with this invention, uponimplantation, undergo biological remodeling. They serve as a rapidlyvascularized matrix for support and growth of new endogenous connectivetissue. When used as a tissue graft material stomach submucosa has beenfound to be trophic for host tissues with which it is attached orotherwise associated in its implanted environment. The graft materialhas been found to be remodelled (resorbed and replaced with autogenousdifferentiated tissue) to assume the characterizing features of thetissue(s) with which it is associated at the site of implantation.

Applicants anticipate that stomach submucosa can be used for tendon andligament replacement and repair as has been previously described forintestinal submucosa. Furthermore, for tendon and ligament replacementapplications, and other connective tissue repair applications thestomach submucosa material will typically be conditioned, as describedin U.S. Pat. No. 5,275,826 (the disclosure of which is expresslyincorporated herein by reference) to alter the viscoelastic propertiesof the submucosal tissue and reduce the strain of the originallyisolated stomach submucosal tissue. The term strain as used hereinrefers to the maximum amount of tissue elongation before failure of thetissue, when the tissue is stretched under an applied load. It isexpressed as a percentage of the length of the tissue before loading. Inaccordance with one embodiment stomach submucosa delaminated from boththe luminal portion of the tunica mucosa and the smooth muscle layers ofthe muscularis externa of a stomach of a warm blooded vertebrate isconditioned to have a strain of no more than 20%. The submucosal tissueis conditioned by stretching, chemically treating, enzymaticallytreating or exposing the tissue to other environmental factors.

In one embodiment the strips of stomach submucosa tissue are conditionedby stretching the submucosa tissue longitudinally to form a graftconstruct having a length longer than the length of the stomachsubmucosa from which the graft construct was formed, and moreparticularly, by stretching in a longitudinal or lateral direction sothat the strips of intestinal submucosa tissue have a strain of no morethan 20%.

One method of "conditioning" the tissue by stretching involves applyinga given load to the stomach submucosa for three to five cycles. Eachcycle consists of applying a load to the graft material for fiveseconds, followed by a ten second relaxation phase. Three to five cycleswill produce a stretch-conditioned graft material with reduced strain.The graft material does not return to its original size; it remains in a"stretched" dimension. For example, a stomach submucosa segment can beconditioned by suspending a weight from said segment, for a period oftime sufficient to produce a tissue having a strain of less than 20%,for example, about 10 to about 20%. Optionally, the graft material canbe preconditioned by stretching in the lateral dimension. The graftmaterial exhibits similar viscoelastic properties in the longitudinaland lateral dimensions.

The graft segment is then formed, in accordance with one embodiment, ina variety of shapes and configurations, for example, to serve as aligament or tendon replacement or a patch for a broken or severed tendonor ligament. The segment can be shaped and formed to have a layered oreven a multilayered configuration with at least the opposite endportions and/or opposite lateral portions being formed to have multiplelayers of the graft material to provide reinforcement for attachment tophysiological structures, including bone, tendon, ligament, cartilageand muscle. In ligament replacement applications, opposite ends can beattached using standard surgical techniques to first and second bones,respectively, with the bones typically being articulated as in the caseof a knee joint.

The end portions of the stomach submucosa composition can be formed,manipulated or shaped to be attached, for example, to a bone structurein a manner that will reduce the possibility of graft tearing at thepoint of attachment. Preferably the material can be folded or partiallyeverted to provide multiple layers for gripping, for example, withspiked washers or staples.

Alternatively, the stomach submucosa composition may be folded back onitself to join the end portions to provide a first connective portion tobe attached, for example, to a first bone and a bend in the intermediateportion to provide a second connective portion to be attached to asecond bone articulated with respect to the first bone. For example, oneof the end portions may be adapted to be pulled through a tunnel in, forexample, the femur and attached thereto, while the other of the endportions may be adapted to be pulled through a tunnel in the tibia andattached thereto to provide a substitute for the natural cruciateligament, the segment being adapted to be placed under tension betweenthe tunnels to provide a ligament function, i.e., a tensioning andpositioning function provided by a normal ligament.

Multiple strips/pieces of stomach submucosa can be overlapped andcompressed, under conditions allowing dehydration of the tissue, to fusethe strips/pieces of the stomach submucosal tissue into a unitarymulti-laminate construct. It is anticipated that the process for formingthe intestinal submucosa constructs described in U.S. patent applicationSer. No. 08/418,515 (the disclosure of which is expressly incorporatedherein by reference) are applicable to stomach submucosa and can be usedto form multi-laminate constructs of stomach submucosa.

The present stomach submucosa compositions may be sterilized usingconventional sterilization techniques including tanning withglutaraldehyde, formaldehyde tanning at acidic pH, ethylene oxidetreatment, propylene oxide treatment, gas plasma sterilization, gammaradiation, and peracetic acid sterilization. A sterilization techniquewhich does not significantly weaken the mechanical strength andbiotropic properties of the graft is preferably used. For instance, itis believed that strong gamma radiation may cause loss of strength inthe graft material. Because one of the most attractive features of thegraft constructs formed from stomach submucosal tissue is their abilityto induce host-remodelling responses, it is desirable not to use asterilization approach which will detract from that property. Preferredsterilization techniques include exposing the graft to peracetic acid,low dose gamma irradiation (≦2.5 mRad) and gas plasma sterilization;peracetic acid sterilization being the most preferred method. Typically,after the tissue graft composition has been sterilized, the compositionis wrapped in a non-porous plastic wrap and sterilized again usingethylene oxide or gamma irradiation sterilization techniques.

The stomach submucosal tissue compositions of the present invention canalso be used in accordance with this invention in a method andcomposition for supporting the proliferation and inducingdifferentiation of eukaryotic cells cultured in vitro. Procedures forutilizing submucosal tissue for the in vitro culturing of cells isdescribed in U.S. patent application Ser. No. 08/386,452, the disclosureof which is expressly incorporated herein. Generally the methodcomprises the step of contacting eukaryotic cells, in vitro, with avertebrate stomach submucosa-derived matrix under conditions conduciveto eukaryotic cell growth.

In one embodiment a eukaryotic cell culture substrate is preparedcomprising submucosal tissue. The submucosal tissue can be combined withvarious nutrients, growth factors, minerals and salts know by thoseskilled in the art to be important in cell culture procedures. The term"contacting" as used herein with reference to cell culture is intendedto include both direct and indirect contact, for example in fluidcommunication, of the submucosal tissue and the cultured cells. The term"conditions conducive to eukaryotic cell growth" as used herein refersto the environmental conditions, such as sterile technique, temperatureand nutrient supply, that are considered optimal for eukaryotic cellgrowth under currently available cell culture procedures. Althoughoptimum cell culture conditions used for culturing eukaryotic cellsdepend somewhat on the particular cell type, cell growth conditions aregenerally well known in the art.

Furthermore, stomach submucosal tissue can be combined with currentavailable cell culture media to enhance the effectiveness of such mediato induce growth, proliferation and differentiation of various celltypes.

EXAMPLE 1 Preparation of Stomach Submucosal Tissue

The tissue graft material of this invention is prepared in accordancewith the following steps:

The stomach is first removed from the animal source by cutting theesophagus and small intestine at their respective entrance and exitpoints on the stomach. Any excess mesentery tissue or fat is removedfrom the stomach and the contents of the stomach are emptied and anyremaining residues are removed from the inside of the stomach by rinsingwith running tap water. The stomach is then everted to expose the insidelayers of the stomach. The portions of the stomach that begin to formthe entrance or exit points of the stomach are removed. The stomach istypically left whole, however the stomach can also be cut and flattenedprior to removal of unwanted tissues.

The luminal surface of the stomach is subject to abrasion using thehandle portion of a pair of scissors or hemostats to scrape off theinner layers of the stomach including at least the luminal portion ofthe tunica mucosa. A thin residual layer will remain at this point. Ifthe tissue was left whole, the stomach tissue is everted again to returnthe luminal surface of the stomach to the interior of the graftconstruct. A small cut is then made in the exterior muscle fiber layer.The muscle layers are then delaminated from the submucosal tissuethrough the use of a pair of scissors or hemostat to enlarge the cut inthe muscle and scrape off the muscle layers. The remaining tissue iseverted again to place the luminal side on the exterior of the tissuegraft. The luminal surface is scraped to remove the remaining insideresidue which has a brownish color. The stomach tissue is scraped untilthe tissue appears pinkish-white in color.

During the preparation of the stomach tissue care is taken to keep thetissue moist by periodically hydrating the tissue with water. Thestomach submucosal tissue is rinsed in running tap water forapproximately two hours to remove any blood or loose tissue scrapingsand lyse and remaining cells. After rinsing the tissue should appearwhite, if the tissue remains pinkish in color the tissue is rubbed underwater until the tissue appears white. After rinsing is complete excesswater is removed by ringing the tissue by hand or the use of mechanicalringers. The tissue is then stored in liquid nitrogen at -80° C.

EXAMPLE 2 Mechanical Properties of Stomach Submucosa

The mechanical properties of stomach submucosa biomaterial has beenevaluated using two different bench top tests: the "wet diaphragm bursttest" and the "ball burst test".

The wet diaphragm burst test involves the placement of the material inan aperture to form a "diaphragm" comprising submucosa tissue held by asurrounding ring. Water pressure is applied to one side of the materialuntil the point of failure. The accumulated data from 10 separate testsamples of each material is presented in Table 1, comparing the wetdiaphragm burst test of intestinal submucosa, urinary bladder submucosa,and stomach submucosa. The terms "in medium" and "frozen" indicate themethod by which the materials were stored subsequent to harvesting andprior to testing.

                                      TABLE 1                                     __________________________________________________________________________    Wet Diaphragm Burst Test (Pressure, in Kg/cm.sup.2)                                       Urinary                                                                              Urinary                                                         Intestinal                                                                           Bladder                                                                              Bladder                                                                              Stomach                                                                              Stomach                                           Submucosa in                                                                         Submucosa in                                                                         Submucosa                                                                            Submucosa in                                                                         Submucosa                                    Sample #                                                                           Medium Medium Frozen Medium Frozen                                       __________________________________________________________________________    1    1.173  0.107  0.391  2.264  0.723                                        2    0.992  0.141  0.548  2.011  1.130                                        3    0.880         0.439  2.148  0.905                                        4    1.350         0.343  2.444  1.234                                        5    1.313         0.455  2.382  1.094                                        6    0.692         0.574  2.463  1.403                                        7    0.810         0.602  2.474  0.959                                        8    0.987         0.354  2.882  1.198                                        9    0.942         0.498  2.275  0.703                                        10   0.827         0.275  2.415  1.603                                        Average                                                                            0.990  0.124  0.448  2.376  1.095                                        Stan.                                                                               0.2196                                                                               0.0239                                                                               0.0108                                                                               0.2329                                                                               0.2850                                      Deviation                                                                     __________________________________________________________________________

The strength of stomach submucosal tissue grafts can also be determinedthrough the use of a material testing system (MTS) tensile tester. Thestomach submucosal tissue sheet is secured within a circular clamp(specimen clamp) to provide uniform distribution of the stress throughout the tissue sheet. The handle of the specimen clamp is lifted to itstopmost position so that the jaws of the clamp are able to accept thetest specimen. The submucosal tissue construct is cut to fit thespecimen clamp, the aperture of the clamp having a diameter of one andnine sixteenths (4 cm). Approximately 1.3-1.5 cm of excess materialshould be included around the perimeter of the test specimen to ensuresufficient clamping area. The submucosal tissue is placed in jaws of theclamp and secured, the clamp force being controlled by thumbwheel meanslocated on the top clamp.

The initial fixture level is set so that the top of the steel ball islocated immediately under the plane the test specimen. The metal ballforced up against the clamped submucosal tissue at a controlled rateutilizing a tensile tester software interface to control and measure theforce placed on the test specimen. The force is increased until failureof the specimen occurs. Failure is defined as the maximum load whichcorresponds to the first appearance of the ball through visiblenon-natural discontinuities in the plane of the specimen. In the casethat the topmost position of the fixture is reached prior to failure,the software limits will engage and discontinue the test. The peak loadvalue displayed on the Microprofiler 458.01 is recorded and the specimenis removed. Table 2 presents the accumulated data of the ball burst testcomparing three different sources of submucosa: intestinal submucosa,urinary bladder submucosa and stomach submucosa. These resultsdemonstrate that stomach submucosa is the strongest of these threedifferent biomaterials.

                  TABLE 2                                                         ______________________________________                                        Ball burst test (maximum load in Kg)                                                       Urinary                                                          Intestinal Submucosa                                                                       Bladder Submucosa                                                                           Stomach Submucosa                                  ______________________________________                                        2.05 ± 0.05                                                                             1.57 ± 0.05                                                                              8.62 ± 3.06                                     ______________________________________                                    

EXAMPLE 3 In-Vivo Remodeling

Sections of stomach submucosa, were used as a scaffold to replaceportions of excised urinary bladder in the dog. Two experiments weredone. One animal was sacrificed after three weeks and the second animalwas sacrificed after six weeks. The morphologic evaluation of theremodeled tissues showed growth of transitional epithelium over thesurface of the stomach submucosa scaffold and the presence of smoothmuscle cells within the bioscaffold. Because there was no definitive wayof identifying the presence or absence of remaining stomach submucosa,the degree and/or extent of degradation of stomach submucosa cannot bedetermined from these studies.

EXAMPLE 4 In-vitro Cell Growth Properties of Stomach Submucosa

The ability of stomach submucosa to serve as an extracellular matrix tosupport in-vitro cell growth was tested by applying several cell typesto the stomach submucosal tissue surface under standard cell cultureconditions. The cell types tested included 3T3 fibroblasts, intestinalepithelium cells and FR (fetal rat) mesenchymal cells. All three celltypes showed the ability to proliferate readily upon this extracellularmatrix without the addition of the supplements that would be needed togrow these cells on a plastic surface. Therefore, it can be concludedthat the material contains necessary structure and composition"nutrients" to serve as a cell culture substrate for supporting cellgrowth.

What is claimed is:
 1. A composition capable of inducing the formationof endogenous tissue when implanted at a site in need of endogenoustissue growth comprising stomach submucosa delaminated from both theluminal portion of the tunica mucosa and the smooth muscle layers of themuscularis externa of a stomach of a warm blooded vertebrate.
 2. Thecomposition of claim 1 wherein the stomach submocosa is fluidized. 3.The composition of claim 1 wherein the stomach submucosa is digestedwith an enzyme for a period of time sufficient to solubilize the stomachsubmucosa and provide a substantially homogenous solution.
 4. Thecomposition of claim 2, wherein the stomach submucosais dried and inpowder form.
 5. The composition of claim 1 formed into a cylinder havinga predetermined luminal diameter and sutured along the length of thecylinder.
 6. The composition of claim 1 conditioned to reduce the strainof the isolated stomach submucosa for use as a connective tissuesubstitute.
 7. The composition of claim 6 wherein the stomach submucosais conditioned by stretching to produce a graft construct longer in atleast one dimension than the stomach submucosa from which it is formed.8. The use of stomach submucosa for the preparation of a tissue graftconstruct useful for inducing endogenous tissue growth when implanted inwarm-blooded vertebrates, said stomach submucosa being delaminated fromboth the luminal portion of the tunica mucosa and the smooth musclelayers of the muscularis externa of a stomach of a warm bloodedvertebrate.
 9. The use according to claim 8 wherein the stomachsubmucosa is in fluidized form.
 10. The use according to claim 8 whereinthe stomach submucosa is in powder form.
 11. A method for inducing theformation of endogenous tissue at a site in need of endogenous tissuegrowth in a warm blooded vertebrate, said method comprising contactingsaid site with a graft composition comprising stomach submucosa in anamount effective to induce endogenous tissue growth at the site thecomposition is administered.
 12. The method of claim 11, wherein thegraft composition is fluidized and is administered by injection into thewarm-blooded vertebrate.
 13. The method of claim 12, wherein the graftcomposition is administered by surgically implanting the compositioninto the warm-blooded vertebrate.